The purification, concentration and/or size-based separation of target biomolecules within and from a sample is important to a number of molecular biology and biochemistry based techniques. For example, desalting of peptide solutions prior to spectral analysis is critical to the sensitivity and validity of the analysis. These aims are especially true with regard to smaller volume bio-molecule containing sample sizes, i.e., microliters of sample solution, or for more dilute bio-molecule containing samples, i.e., samples having less than about 10 μg/ml of target bio-molecule in solution.
Conventional purification, concentration and sized-based separation techniques have focused on various filtration and chromatographic techniques. For example, samples are positioned or placed on a pre-set filter membrane within a microfuge tube, the filter membrane defining pore sizes that prevent the target bio-molecule from washing through the filter. Centrifugation of the microfuge tube forces smaller molecules within the sample to pass through the pores of the filter, while retaining the larger (than the pores) size molecules to remain on the filter. This process ultimately concentrates, purifies and separates target biomolecules from other smaller constituents. However, these ultrafiltration techniques often require long centrifugation or filtration steps, to move the sample through the filter pores. Furthermore, ultrafiltration membranes are very sensitive to viscosity, often resulting in clogging problems.
Recently, a polymer matrix based plug has been developed for separating and concentrating target molecules from a sample. The plug is prepared by casting the polymer and sorptive bead material into an appropriate housing, for example a pipette tip. See, e.g., U.S. Pat. No. 6,200,474. The homogenous polymer matrix formed by the casting process provides a continuous polymer material capable of entrapping sorptive type particles, the entire plug acting to concentrate, purify and separate target biomolecules. Unfortunately, however, polymer matrix-based techniques require solvent based casting techniques, which often require time, harmful solvents, and specialized equipment for the fabrication of housing containing plugs.
Against this backdrop the following disclosure is provided.